A high-content AlphaScreen™ identifies E6-specific small molecule inhibitors as potential therapeutics for HPV+ head and neck squamous cell carcinomas

Advances in Pharmacological and Pharmaceutical Sciences

Krstenansky

Duerksen-Hughes

2021

Self Cite

The COVID-19 pandemic that began in late 2019 continues with new challenges arising due to antigenic drift as well as individuals who cannot or choose not to take the vaccine. There is therefore an urgent need for additional therapies that complement vaccines and approved therapies such as antibodies in the fight to end or slow down the pandemic. SARS-CoV-2 initiates invasion of the human target cell through direct contact between the receptor-binding domain of its Spike protein and its cellular receptor, angiotensin-converting enzyme-2 (ACE2). The ACE2 and S1 RBD interaction, therefore, represents an attractive therapeutic intervention to prevent viral entry and spread. In this study, we developed a proximity-based AlphaScreen™ assay that can be utilized to quickly and efficiently screen for inhibitors that perturb the ACE2 : S1 RBD interaction. We then designed several peptides candidates from motifs in ACE2 and S1 RBD that play critical roles in the interaction, with and without modifications to the native sequences. We also assessed the possibility of reprofiling of candidate small molecules that previously have been shown to interfere with the viral entry of SARS-CoV. Using our optimized AlphaScreen™ assay, we evaluated the activity and specificity of these peptides and small molecules in inhibiting the binding of ACE2 : S1 RBD. This screen identified cepharanthine as a promising candidate for development as a SARS-CoV-2 entry inhibitor.

Section: Methodsmentioning

confidence: 99%

ACE2 : S1 RBD Interaction-Targeted Peptides and Small Molecules as Potential COVID-19 Therapeutics

Advances in Pharmacological and Pharmaceutical Sciences

Krstenansky

Duerksen-Hughes

2021

Self Cite

“…We previously identified an E6 specific inhibitor, GA-OH, from a high content screen and have shown that it increases caspase 8 and p53 levels in HPV + cells (25). Given that expression of wildtype p53 has been identified as a critical biomarker for radiosensitivity in HNSCC and the role caspase 8 plays in apoptosis, we first investigated how GA-OH modulates radiosensitivity of 2 HPVand 3 HPV + cell lines.…”

Section: Effect Of Ga-oh On Radio-cytotoxicity As Compared To Cisplat...mentioning

confidence: 99%

“…We previously discovered an E6 inhibitor, GA-OH, and demonstrated that it can rescue pro-apoptotic molecules, including p53, and promote apoptosis (25). We therefore tested the efficacy of GA-OH in combination with radiation to evaluate radio-potentiation.…”

Section: Introductionmentioning

confidence: 99%

GA-OH enhances the cytotoxicity of photon and proton radiation in HPV+ HNSCC cells

Bertucci²,

Vazquez³

et al. 2023

Front. Oncol.

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IntroductionTreatment-related toxicity following either chemo- or radiotherapy can create significant clinical challenges for HNSCC cancer patients, particularly those with HPV-associated oropharyngeal squamous cell carcinoma. Identifying and characterizing targeted therapy agents that enhance the efficacy of radiation is a reasonable approach for developing de-escalated radiation regimens that result in less radiation-induced sequelae. We evaluated the ability of our recently discovered, novel HPV E6 inhibitor (GA-OH) to radio-sensitize HPV+ and HPV- HNSCC cell lines to photon and proton radiation.MethodsRadiosensitivity to either photon or proton beams was assessed using various assays such as colony formation assay, DNA damage markers, cell cycle and apoptosis, western blotting, and primary cells. Calculations for radiosensitivity indices and relative biological effectiveness (RBE) were based on the linear quadratic model.ResultsOur results showed that radiation derived from both X-ray photons and protons is effective in inhibiting colony formation in HNSCC cells, and that GA-OH potentiated radiosensitivity of the cells. This effect was stronger in HPV+ cells as compared to their HPV- counterparts. We also found that GA-OH was more effective than cetuximab but less effective than cisplatin (CDDP) in enhancing radiosensitivity of HSNCC cells. Further tests indicated that the effects of GA-OH on the response to radiation may be mediated through cell cycle arrest, particularly in HPV+ cell lines. Importantly, the results also showed that GA-OH increases the apoptotic induction of radiation as measured by several apoptotic markers, even though radiation alone had little effect on apoptosis.ConclusionThe enhanced combinatorial cytotoxicity found in this study indicates the strong potential of E6 inhibition as a strategy to sensitize cells to radiation. Future research is warranted to further characterize the interaction of GA-OH derivatives and other E6-specific inhibitors with radiation, as well as its potential to improve the safety and effectiveness of radiation treatment for patients with oropharyngeal cancer.

“…In addition to these inhibitors, we recently discovered another inhibitor with a nonflavonoid scaffold (30 hydroxygambogic acid, or GA-OH) as a potential therapeutic for HPV + cancer cells. We demonstrated that GA-OH significantly inhibited the survival and growth of HPV + cell lines and displayed higher potency than did flavonols such as myricetin [ 73 ]. In the future, we hope to study whether this inhibitor will potentiate the activity of standard therapies in HPV-associated cells and how it interacts with E6.…”

Section: Therapeutic Targeting Of E6 Ppismentioning

confidence: 99%

PPI Modulators of E6 as Potential Targeted Therapeutics for Cervical Cancer: Progress and Challenges in Targeting E6

Duerksen-Hughes

2021

Molecules

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Advanced cervical cancer is primarily managed using cytotoxic therapies, despite evidence of limited efficacy and known toxicity. There is a current lack of alternative therapeutics to treat the disease more effectively. As such, there have been more research endeavors to develop targeted therapies directed at oncogenic host cellular targets over the past 4 decades, but thus far, only marginal gains in survival have been realized. The E6 oncoprotein, a protein of human papillomavirus origin that functionally inactivates various cellular antitumor proteins through protein–protein interactions (PPIs), represents an alternative target and intriguing opportunity to identify novel and potentially effective therapies to treat cervical cancer. Published research has reported a number of peptide and small-molecule modulators targeting the PPIs of E6 in various cell-based models. However, the reported compounds have rarely been well characterized in animal or human subjects. This indicates that while notable progress has been made in targeting E6, more extensive research is needed to accelerate the optimization of leads. In this review, we summarize the current knowledge and understanding of specific E6 PPI inhibition, the progress and challenges being faced, and potential approaches that can be utilized to identify novel and potent PPI inhibitors for cervical cancer treatment.